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Purification of two murine monoclonal antibodies of the IgM class by hydroxylapatite chromatography and gel filtration
Author(s) -
Henniker A. J.,
Bradstock K. F.
Publication year - 1993
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130070302
Subject(s) - hydroxylapatite , chemistry , monoclonal antibody , chromatography , size exclusion chromatography , filtration (mathematics) , antibody , biochemistry , immunology , enzyme , biology , statistics , mathematics
A two‐step method using hydroxylapatite chromatography and gel filtration is described for the purification of two murine monoclonal antibodies of the IgM class. Ascites fluid from each hybridoma was diluted in sodium phosphate buffer (0.01 M, pH 6.8), loaded onto a hydroxylapatite column and eluted with a stepwise sodium phosphate gradient. The immunoreactive protein peaks were concentrated and subjected to gel filtration using either Sephadex G‐200 or Sephacryl S‐200HR. The biological activity of the end‐products was confirmed by complement lysis assay and by indirect immunofluorescence and flow cytometry. The purity of the end‐products as assessed by SDS polyacrylamide gel electrophoresis and densitometry was at least 90%. The methods described produced immunoreactive material with a high level of purity. The procedure for each antibody was reproducible and provides a reliable method for purification of monoclonal antibodies of the IgM class.